Evidence That Aspartic Acid 301 Is a Critical Substrate-Contact Residue in the Active Site of Cytochrome P450 2D6

Abstract

Model building studies have intimated a role for aspartic acid 301 in the substrate binding of cytochrome P450 2D6 (CYP2D6). We have tested this hypothesis by generating a range of CYP2D6 mutants substituting a variety of amino acids at this site. The mutant proteins, which included substitution with a negatively charged glutamic acid residue or neutral asparagine, alanine, or glycine residues, were expressed in Saccharomyces cerevisiae. In addition, a mutant where aspartic acid 301 was deleted was also tested. All the mutants expressed approximately equivalent amounts of recombinant apoprotein and, apart from the alanine 301 and the aspartic acid 301 deletion mutants, gave carbon monoxide difference spectra of similar magnitude to the wild type. In the cases of the alanine and deletion mutants, the amount of holoprotein was significantly reduced or absent relative to the amount of apoprotein, indicating restricted heme incorporation. The glutamic acid mutant was shown to have similar catalytic properties to the wild type enzyme toward the substrates debrisoquine and metoprolol; however, some differences in regioselectivity and ligand binding were observed. The mutants containing neutral amino acids at position 301 exhibited marked reductions in catalytic activity. At low substrate concentrations little, if any, activity toward debrisoquine and metoprolol was measured. However, at a higher substrate concentration (2 mM) some activity was observed (about 10-20% of wild type levels). Consistent with the above findings, the debrisoquine-induced spin changes in the mutant proteins were markedly reduced. These data collectively demonstrate that aspartic acid 301 plays an important role in determining the substrate specificity and activity of CYP2D6 and provide experimental evidence supporting the role of this amino acid in forming an electrostatic interaction between the basic nitrogen atom in CYP2D6 substrates and the carboxylate group of aspartic acid 301.

Synergistic Use of Compound Properties and Docking Scores in Neural Network Modeling of CYP2D6 Binding: Predicting Affinity and Conformational Sampling

“…Positively charged amines on ligands (colored blue) can be seen at various regions in the binding site, although proximity to Asp301 and Phe120 is seen for some of the higher affinity ligands (Figure 9), as expected. 18,19 Interestingly, the cluster of 82 ligand poses assumes a U-shape, with the curvature apparently caused by steric interference from Leu484. This residue, along with Phe120, creates a 6 Å constriction in the binding site (Figure 9), defining a hydrophobic wall for ligands.…”

Assessment of Arginine 97 and Lysine 72 as Determinants of Substrate Specificity in Cytochrome P450 2c9 (Cyp2c9)

“…These results find a precedent in recent data obtained for CYP2D6. D301 of CYP2D6 was believed to be responsible for interaction with substrate based on site-directed mutagenesis data using enzyme expressed in yeast (Ellis et al, 1995). However, D301 mutants appear to be unstable or poorly expressed in the bacterial system (Hanna et al, 2001), and recent data suggest that D301 may be involved in maintaining the structural integrity of the enzyme by stabilizing the B-C loop (Guengerich et al, 2002;Kirton et al, 2002;Paine et al, 2002).…”

Inactivation of Rat Cytochrome P450 2D Enzyme by a Further Metabolite of 4-Hydroxypropranolol, the Major and Active Metabolite of Propranolol.

“…The rat enzyme may share this property of selectivity for compounds containing cationic (at physiological pH) centers 31) so that amine substrates commonly have K m values in the micromolar range. 16,28,32,33) Similar to CYP2D6, there is an aspartic acid residue at position 304 of rat CYP2D2 of which the carboxyl group can trap a heteroatom of its substrates (Fig. 7).…”

“…[25][26][27] Although inactivity of 1,4-NQ would appear to refute the notion of a quinone-based inactivation process, a more subtle process occurring within the active site of the enzyme may account for the lack of effect of 1,4-NQ and 1,4-DHN. The substrate binding site of CYP2D6, the human form of CYP2D, contains an anionic center to which the cationic form of the substrate binds 4,28) at 5 to 7 Å 29,30) from the oxidation site. The rat enzyme may share this property of selectivity for compounds containing cationic (at physiological pH) centers 31) so that amine substrates commonly have K m values in the micromolar range.…”